Secreted Proteases from Dermatophytes

11046_2008_9105_166_5-web 285..294Michel Monod
Received: 15 October 2007 / Accepted: 30 January 2008 / Published online: 14 May 2008
Springer Science+Business Media B.V. 2008
Abstract Dermatophytes are highly specialized
pathogenic fungi that exclusively infect the stratum
corneum, nails or hair, and it is evident that secreted
proteolytic activity is important for their virulence.
Endo- and exoproteases-secreted by dermatophytes are
similar to those of species of the genus Aspergillus.
However, in contrast to Aspergillus spp., dermato-
phyte-secreted endoproteases are multiple and are
members of two large protein families, the subtilisins
(serine proteases) and the fungalysins (metalloproteas-
es). In addition, dermatophytes excrete sulphite as a
reducing agent. In the presence of sulphite, disulphide
bounds of the keratin substrate are directly cleaved to
cysteine and S-sulphocysteine, and reduced proteins
become accessible for further digestion by various
endo- and exoproteases secreted by the fungi. Sulphi-
tolysis is likely to be an essential step in the digestion of
compact keratinized tissues which precedes the action
of all proteases.
Abbreviations
Scp Serine carboxypeptidase
fungi which are the most common agents of super-
ficial mycoses [1]. They are not opportunists, but true
pathogenic fungi infecting stratum corneum, nails or
hair of healthy individuals. During infection, hard
keratin tissues have to be digested into short peptides
and amino acids in order to be assimilated via
transporters. The aim of this paper is to briefly review
different properties of the proteases secreted by
dermatophytes in comparison to other pathogenic
fungi, and describe the different steps of keratinized
tissue digestion.
M. Monod (&)
Mycologie, BT422, Centre Hospitalier Universitaire
Vaudois, 1011 Lausanne, Switzerland
DOI 10.1007/s11046-008-9105-4
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proteolytic enzyme and peptide hydrolase. The
proteases include all enzymes that catalyse the
cleavage of the peptide bonds (CO–NH) of proteins,
digesting these proteins into peptides or free amino
acids. The classification and the nomenclature of
proteases can be found together with information
about them in the Handbook of proteolytic enzymes
[2] and in the MEROPS database accessible at
http://merops.sanger.ac.uk/. The proteases are ini-
tially classified following their mode of action and
their active sites. Aspartic, cysteine, glutamic, me-
tallo, serine and threonine proteases as well as
proteases with unknown catalytic mechanism are
recognized. Each protease is then assigned to a family
that is a set of homologous enzymes (Table 1). These
families are identified by a capital letter representing
the catalytic type of the peptidases they contain,
together with a unique number. Subfamilies are
labelled with a second capital letter (for instance, S9B
and S9C in the serine proteases, Table 1). Different
families that are thought to have a common origin are
grouped into a clan.
proteases (or endopeptidases) and exoproteases (or
exopeptidases). The endoproteases cleave peptide
bonds internally within a polypeptide. The exopro-
teases cleave peptide bonds only at the N- or the
C-terminus of polypeptide chains.
or signal peptide [3] is necessary for entering the
secretory pathway by transporting the protein across
the membrane of the endoplasmic reticulum [4]. Like
in bacteria, the further N-terminal extension called the
propeptide (30–250 amino acids in length) has been
found to be essential and specific in assisting the correct
folding and the secretion of its associated protein [5, 6].
Upon completion of folding, the propeptide is removed
by an autoproteolytic or an exogenous proteolytic
reaction to generate the active enzyme.
Dermatophyte-Secreted Endoproteases
secrete proteolytic activity in vitro when grown in a
medium containing protein as sole nitrogen source
Table 1 Secreted endo- and exoproteases from T. rubrum and other dermatophytes
Clan Protease family or subfamily Identifiera Genesb Proteasesc References
Endoproteases
MA M36 (Fungalysins) M36.001 MEP1–MEP5 Mep1, Mep3, Mep4 [9, 10, 12, 37,
unpublished results]
Exoproteases
S9C (Dipeptidyl peptidases) S09.012 DPPV DppV [14]
MH M28E M28.006 LAP1 Lap1 [14]
M28A (Aminopeptidases) M28.001 LAP2 Lap2 [14]
MC M14 (Carboxypeptidases) Unassigned MCPA, MCPB McpA, McpB [Unpublished]d
SC S10 (Carboxypeptidases) S10.016 SCPA, SCPB ScpA, ScpB [Unpublished]e
a With the same identifier is a set of proteins all of which display a particular kind of peptidase activity, and are closely related in
sequence. The identifier is made of the family name and an arbitrary three-digit serial number [2] b Nomenclature adopted for T. rubrum and M. canis genes [10–14] c T. rubrum proteases or orthologues of other dermatophyte species which were detected in culture supernatants and/or obtained as
recombinant protein d Genbank accession number DQ778058 (MCPA) and DQ786567 (MCPB) e Genbank accession number AY497023 (SCPA) and AY497022 (SCPB)
286 Mycopathologia (2008) 166:285–294
amino acids repress the genes coding for secreted
proteases. There are many works reporting the
isolation and characterization of one or two secreted
proteases from an individual species of dermatophyte
[8, 9, 15–28] (Table 2). These enzymes were often
described as keratinases without paying attention to
the cornified cell envelope made of other components.
Keratinized tissues such as the epidermis, nails and
hair are not only made of keratins, but also of an
insoluble network made of different cross-linked
proteins such as involucrin, loricrin and small proline
rich proteins which form the cornified cell envelope
[29–32]. With the exception of a Microsporum canis
31.5-kDa serine protease [8] belonging to the subtil-
isin family and a 43.5-kDa metalloprotease [9], there
were no reported amino acid sequences of the N-
terminus of these proteins to further identify and
characterize the isolated extracellular enzymes. The
isolated M. canis serine protease and metalloprotease
were subsequently called Sub3 (for subtilisin 3) and
Mep3 (for metalloprotease 3), respectively, after their
genes had been cloned and expressed in Pichia
pastoris. The N-terminus sequences of two different
peptides of 34 and 48 kDa were reported for one
M. canis keratinase called ‘‘Ekase’’ [27]. However,
they are identical to the N-terminus sequences of Sub3
and Mep3, respectively, and therefore, the ‘‘Ekase’’
corresponds to a mixture of two proteases.
The growth of dermatophytes is rather slow and it is
difficult to obtain enough material for the purification
of native proteins in sufficient quantities for further
characterization. Therefore, a reverse genetic
approach (from genes to proteins), which allowed the
production of individual proteases in substantial
amounts, was chosen to investigate proteolytic activity
of different dermatophyte species. This experimental
approach also avoided the problem of purification
since there would be no contamination from other
proteases present in the culture supernatant, as it
happens when these fungi are grown in a protein
medium. Briefly, dermatophytes were shown to pos-
sess a genome encoding a battery of secreted proteases
similar to that of Aspergillus species (Table 1).
However, five MEP and seven SUB genes isolated in
Trichophyton rubrum and in other dermatophytes
Table 2 Secreted proteases which were purified from dermatophytes culture supernatants, and subsequently characterized
Dermatophyte species Molecular mass (kDa) References Comments
T. rubrum 34.7 [15] Subtilisin (MM and PMSF inhibition)
T. rubrum 36 [16] From SDS-PAGE, likely a Sub
T. rubrum 44 [16] From SDS-PAGE, likely a Mep
T. rubrum 27 [17]
T. mentagrophytes 48 [18]
T. mentagrophytes 440 [19]
T. mentagrophytes 20.3 [19]
T. mentagrophytes 38–41 [20] Differs from other proteases by its activity
at acidic pH
T. mentagrophytes var erinacei 33 [21] Subtilisin (MM and PMSF inhibition)
T. mentagrophytes (various
strain)
[22]
T. vanbreuseghemii 37 [23] From SDS-PAGE and PMSF inhibition, likely Sub7
[37]
M. canis 45 [24–26] Likely a Mep (MM), but inhibited by PMSF
M. canis 48, 34, 31.5 kDa fragments
(under the name of
The 48-kDa fragment is M. canis Mep3 [10]
(N-terminal amino acid sequences)
M. canis 33 [28] Likely a Sub (MM and activity at alkaline pH)
M. canis 31.5 [8] Sub3 [11]
M. canis 43.5 [9] Mep3 [10]
Mycopathologia (2008) 166:285–294 287
123
neutral- and alkaline-secreted endoproteases in Asper-
gillus spp. Aspergillus fumigatus has only one gene
(MEP) encoding a secreted fungalysin (MER-
OPS [ M36.001), and two (ALP1 and ALP2)
encoding secreted subtilisins [alkaline proteases 1
(MEROPS [ S08.053) and 2 (MEROPS [ S08.052)]
[33–36; see also the A. fumigatus Af293 genome
(http://www.tigr.org/tdb/e2k1/afu1/)]. Alp1 mep mutants
ALP2 encodes the vacuolar subtilisin which is the
orthologue of the proteinase B in the yeast Saccharo-
myces cerevisiae (MEROPS [ S08.052) [36].
The genome of dermatophyte species so far
encodes the same set of secreted proteases, and the
protein sequence of each orthologue is highly con-
served across species [12, 13, 37]. A phylogenetic
analysis of genomic and protein sequences of metal-
loproteases of the fungalysin family revealed a robust
tree consisting of five main clades, each of them
including a MEP sequence type from each dermato-
phyte species [12]. The tree topology clearly indicated
that the multiplication of MEP genes in dermato-
phytes occurred prior to species divergence. Likewise,
five genes encoding T. rubrum Subs revealed a
specific intron–exon structure [13]. Based on
sequence data and intron–exon structure, a phyloge-
netic analysis of subtilisin genes from T. rubrum and
other dermatophyte species revealed a presumably
ancestral lineage comprising SUB2 orthologues and
Aspergillus ALP1. All other genes encoding secreted
subtilisins (SUB1, SUB3–SUB7) are dermatophyte
specific and seem to have emerged more recently,
through successive gene duplication events [13].
The availability of gene sequences allowed the
identification of proteases on SDS-PAGE gels by
mass spectrometry (MS) [12, 13, 37]. Stained bands
were excised and in-gel digested with trypsin. The
digestion products were analysed by reversed-phase
liquid chromatography-MS/MS. Alternatively, prote-
specific antibodies raised against individual recombi-
nant proteases produced in Pichia pastoris or large
peptides produced in Escherichia coli [37]. Using
both identification methods, several Meps and Subs
were shown to be secreted by dermatophytes in a
protein medium [12, 13, 37]. However, their levels of
secretion differ significantly from one species to
another [37]. For instance, Sub7 is the major protease
secreted by Trichophyton tonsurans. Sub7 is also
secreted in different Trichophyton species, but was
not detected in Trichophyton soudanense culture
supernatants. In contrast Sub6 was found in
T. soudanense culture supernatants, together with
other Subs, but was not detected in culture superna-
tants from other species.
Dermatophyte Subs show an apparent molecular
mass of 30–37 kDa and are not glycosylated (Fig. 1).
As an exception mature Sub6 appears as a 20-kDa
polypeptide chain on SDS-PAGE gels [37]. Derma-
tophyte Subs are made as preproproteins with a
prosequence of about 100 amino acids which is
removed from the mature catalytic domain of the
protease by autoproteolytic activity. In contrast to
metalloproteases of the fungalysin family (see
below), but like other subtilisins, they very efficiently
cleave the synthetic substrate N-Suc-Ala-Ala-Pro-
Phe-p-nitroanilide (pNA) [8, 23]. They are alkaline
kDa
95
66
45
31
Lap2
tant of Trichophyton rubrum (Tru) and Arthroderma benhamiae (Abe). Both dermatophytes were grown in a
medium containing soy proteins as the sole nitrogen and
carbon source. The proteins of 2 ml of culture supernatant were
precipitated using trichloroacetic acid, and resuspended in a
total volume of 10-ll loading buffer. Major secreted peptidases
are annotated. For comparison, T. rubrum recombinant Sub3
(rSub3) produced in P. pastoris is shown in this figure. Ten
microlitres of P. pastoris culture supernatant were loaded. The
9% polyacrylamide SDS-PAGE gel was stained with
Coomassie brilliant blue. Molecular mass markers (M) are
shown on the leftmost lane
288 Mycopathologia (2008) 166:285–294
proteases with optimal activity at pH 7.0–9.0, and are
strongly inhibited by phenyl methyl sulphonyl fluo-
ride (PMSF) and chymostatin.
an apparent molecular mass of 40–48 kDa (Fig. 1).
They are synthesized as preproproteins with a
prosequence of about 240 amino acids which is
removed from the mature catalytic domain of the
protease by autoproteolytic activity as in Subs. The
fungalysins are Zn metalloproteases with a HEXXE
motif in their amino acid sequence [38]. Their
optimum pH of activity is between 7.0 and 8.0.
These neutral proteases are totally inhibited by
phosphoramidon and other chelating agents such as
EDTA and 1–10 phenanthroline, and are insensitive
to PMSF, pepstatin and iodoacetamide.
Dermatophyte-Secreted Exopeptidases
exon structures of the genes encoding these proteases
are similar to the genes coding for orthologues in
A. fumigatus and Aspergillus oryzae. Lap2 is a major
peptidase secreted by dermatophytes (Fig. 1).
Both T. rubrum Lap1 and Lap2 were called leucine
aminopeptidases like Aspergillus spp. orthologues
because of their preference for leucine-7-amido-4-
methylcoumarin (Leu-AMC) as a substrate. How-
ever, these enzymes are able to remove any amino
acid from the N-terminus of a peptide provided that a
proline is not in second position. Lap1 and Lap2 are
metalloproteases which are active between pH 6.5
and 10.5 with a broad optimum peak between pH 7.0
and 9.0 [14]. Lap1 structurally belongs to the M28E
subfamily as do Vibrio and Aeromonas leucyl
aminopeptidases (MEROPS [ M28.002; MER-
belongs to the M28A subfamily like the vacuolar
protease Y of S. cerevisiae (MEROPS [ M28.001)
and the Streptomyces griseus-secreted aminopepti-
dase (MEROPS [ M28.003). Members of the M28A
and M28E subfamilies share low sequence similarity.
However, the amino acid sequences of two Zn++
binding sites are conserved. Like other fungal-
secreted aminopeptidases, T. rubrum Lap1 and
Lap2 were found to be sensitive to different ions.
Like S. griseus aminopeptidase, Lap2 is highly
activated by Co++ [14].
orthologues from Aspergillus spp., are serine prote-
ases with a Ser, Asp, His catalytic triad, and are
members of the S9 family [14]. Both enzymes are
glycoproteins of approximately 90 kDa with about
10 kDa of N-linked carbohydrates. Recombinant
T. rubrum DppIV and DppV are active between pH 6.5
and 10.5 with a broad optimum peak between pH 7.0
and 9.0. Recombinant DppIV efficiently hydrolyses
X-Pro and X-Ala substrates (X-Pro-AMC, X-Pro-
pNA, X-Ala-AMC and X-Ala-pNA, for instance).
Like human CD26 and A. fumigatus DppIV,
T. rubrum DppIV is inhibited by lys-[Z(NO2)]-pyroli-
dide and lys-[Z(NO2)]-thiazolidide. Recombinant
T. rubrum DppV efficiently hydrolyses X-Ala sub-
strates, but is not capable of removing Gly-Pro from
Gly-Pro substrates. Amino acids and tripeptides are
not removed from amino acid and tripeptide sub-
strates by either DppIV or DppV. No specific
inhibitor has been found for fungal DppVs.
Dermatophytes were also found to secrete a
metallocarboxypeptidase (McpA) of the M14A sub-
family in the MEROPS data library which is
homologous to the human pancreatic carboxypepti-
dase A (Table 1, unpublished results). In addition to
McpA, T. rubrum produces two serine carboxypep-
tidases of the S10 family, ScpA and ScpB, related to
the previously characterized carboxypeptidase S1
secreted by A. oryzae in culture supernatant
(Table 1). However, these two carboxypeptidases
are glycophosphatidylinositol-anchored (unpublished
and Antigenic Properties
endo- and exopeptidases were identified in dermato-
phytes (Table 1), and 15 encoded proteases were
found to be secreted in vitro during the growth of
dermatophytes in a protein medium. However, data
are still lacking on proteases like on other hydrolases
(e.g. ceramidases and lipases) secreted during the
infectious process, and on the kinetics of the
Mycopathologia (2008) 166:285–294 289
123
portions of infected hair structures in cats using
specific anti-Sub3 IgG [39]. In addition, M. canis
SUB1, SUB2, SUB3, MEP2 and MEP3 mRNA were
detected by reverse transcriptase-nested polymerase
chain reaction in hair from experimentally infected
guinea pigs [10, 11]. Since dermatophytes grow
exclusively in the stratum corneum, nails or hair
utilizing them as sole nitrogen and carbon sources, it
is reasonable to postulate that during infection the
dermatophytes behave like in a protein medium, and
also secrete different exoproteases. Synergistic action
of endo- and exoproteases allows the degradation of
the keratinized tissues into amino acids and short
peptides that are assimilable via dermatophyte trans-
porters (see below).
both cell-mediated and humoral immune reactions.
Specific lymphoproliferative response towards
[40, 41]. The response was only transient with Mep3.
Antibody response was observed towards Mep3, but
not towards Sub3 except for one out of fourteen
animals. Although being immunogenic, both prote-
ases were not protective against M. canis
experimental infection in guinea pigs in a vaccination
trial [42, 43].
t4, like Trichophyton extracts, elicit either immediate
(IH) or delayed-type (DTH) hypersensitivity skin test
reactions in different individuals [44]. IH is associ-
ated with chronic, low-grade infections and the
presence of IgE antibodies to purified Trichophyton
antigens. In contrast, DTH is associated with highly
inflamed lesions, which are acute and resolve spon-
taneously. Thus, it has been proposed that cell-
mediated immune responses to Trichophyton antigens
confer protection. Tri r2 is the protease Sub6 [13, 37],
and was identified by screening a phage display
library with sera from individuals with high IgE
antibody titers and IH skin test reactions to a
Trichophyton extract [45]. Tri t4 was purified from
T. tonsurans mycelium extract [46], and is the
orthologue of A. fumigatus and T. rubrum DppV
[14, 45]. Skin tests and in vitro T cell proliferation
assays were used to monitor this antigen in the
isolated fractions during the different steps of its
purification [46].
The endo- and exoproteases of many microorganisms
cooperate very efficiently in protein digestion. During
this process, the main function of the former is to
produce a large number of free ends on which the
latter may act. The high keratinolytic activity of T.
rubrum Sub3 and Sub4 suggests that the dermato-
phyte endoproteases play an important role in
virulence. In comparison to proteinase K and subtil-
isin Carlsberg, Sub3 and Sub4 were more active on
keratin azure, but less active on other protein sources,
especially elastin, suggesting a specificity towards
hard keratin substrates [13].
DppIV and DppV) showed activities similar to those
of A. fumigatus orthologues [14]. Large peptides
generated by endoprotease digestion can subse-
quently be digested into amino acids and X-pro
dipeptides in a way similar to that utilized by
A. oryzae and Lactobacillus spp. [47, 48]. Laps degrade
peptides from their N-terminus; however, X-Pro acts
as a stop sequence. In a complementary manner,
these X-Pro sequences can be removed by DppIV,
thus allowing Laps access to the next residue.
Synergistic action of Lap and DppIV to degrade
large peptides seems to be common to many asco-
mycete fungi. In conclusion, dermatophytes like
Aspergillus spp. secrete a battery of endo- and
exoproteases which allow the digestion of a protein
into oligopeptides and free amino acids which are
then assimilable via transporters.
Preceding Proteolytic Activity
by themselves compact keratinous tissues. Proteins of
the cornified cell envelope, in particular loricrin and
small proline rich proteins, contain numerous cyste-
ine residues that form disulphide bridges. This results
in an insoluble proteinaceous complex made of a
network of different cross-linked proteins. Efficient
protein degradation of keratinous tissues by
290 Mycopathologia (2008) 166:285–294
123
simultaneous reduction of the cystine disulphide
bridges, which are an important structural feature of
keratin complexes [13, 49, 50]. Efficient in vitro
degradation of the hair structure of keratin azure by
hydrolytic enzymes was only possible in the presence
of a reducing agent, such as 1% b-mercaptoethanol or
dithiothreitol (DTT).
fungi were shown to excrete sulphite as a reducing
agent [50–52]. In the presence of sulphite, disulphide
bonds of the keratin substrate are directly cleaved to
cysteine and S-sulphocysteine (Fig. 2). The presence
of sulphocysteine was confirmed in human hair
attacked by Microsporum gypseum [53]. This com-
pound was found both in the free form and in
oligopeptides with a molecular mass of about 700–
2,500 Da, the primary products of keratinolysis [52,
54]. As highly specialized fungi, the dermatophytes
are able to utilize free cystine added to a nutrient
broth and excrete sulphite which is a product of the
metabolism of cysteine compounds. In presence of
disulphide bridges, sulphite is immediately bound in
the form of S-sulphocysteine (Fig. 2). Although it
totally inhibits the activity of subtilisins in vitro,
sulphite should not interfere with the activity of these
enzymes secreted by the fungus in vivo. In fact,
sulphite is only detectable in vitro when its amount
exceeds that of cystine [50, 55].
Genes encoding T. rubrum, Arthroderma benh-
amiae and A. fumigatus sulphite efflux pumps
(TruSSU1, AbeSSU1 and AfuSSU1, respectively)
were cloned and expressed…